IMI) in STG neurons. Because IMI is fast and non-inactivating, its peak level does not depend on the temporal properties of neuronal activity. We found, however, that the amplitude and peak time of the proctolin-activated current in LP is frequency dependent. Because frequency affects the rate of voltage change, we measured these currents with voltage ramps of different slopes and found that proctolin activated two kinetically distinct ionic currents: the known IMI, whose amplitude is independent of ramp slope or direction, and an inactivating current (IMI-T), which was only activated by positive ramps and whose amplitude increased with increasing ramp slope. Using a conductance-based model we found that IMI and IMI-T make distinct contributions to the bursting activity, with IMI increasing the excitability, and IMI-T regulating the burst onset by modifying the postinhibitory rebound in a frequency-dependent manner. The voltage dependence and partial calcium permeability of IMI-T is similar to other characterized neuromodulator-activated currents in this system, suggesting that these are isoforms of the same channel. Our computational model suggests that calcium permeability may allow this current to also activate the large calcium-dependent potassium current in LP, providing an additional mechanism to regulate burst termination. These results demonstrate a mechanism for frequency-dependent actions of neuromodulators.In oscillatory circuits, some actions of neuromodulators depend on the oscillation frequency. However, the mechanisms are poorly understood. We explored this problem by characterizing neuromodulation of the lateral pyloric (LP) neuron of the crab stomatogastric ganglion (STG). Many peptide modulators, including proctolin, activate the same ionic current ( Oscillatory neurons respond to synaptic input in complex ways that depend on the polarity, amplitude, and rate of the input, and intrinsic properties of the cell. As a result, neuromodulator inputs that activate voltage-gated ionic currents can have indirect and state-dependent effects. We show that when a target of neuromodulation is a transient ionic current, an additional layer of complexity of the response emerges in which the oscillation frequency and the indirect influence of other ionic currents shape the amplitude and temporal properties of the neuronal response to the modulator.Neuron and network activity is tuned by neuromodulators that influence neuronal excitability and synaptic function, often through G-protein-coupled receptor signaling . An impoICaT), but only when the baseline membrane potential is hyperpolarized (INaP), a transient current such as ICaT, or a hyperpolarization-activated current such as Ih. While persistent currents show little dependence on the history of activity, the voltage and time dependence of channel inactivation makes the contribution of transient currents dependent on oscillation frequency and prior activation , which is crucial for oscillatory activity in pacemaker neurons  of the brain stem respiratory system , and the neurons . However neurons . Because10.1523/ENEURO.0338-21.2021.f1-1Extended Data Figure 1-1Figure 1-1, XLSX file.Depolarization rates for different cycle periods of the unitary realistic LP voltage waveform. Download IMI in all but one pyloric neuron type (I-V) relationship of proctolin-activated currents with voltage ramps of different slopes that correspond to LP depolarization rates at different cycle frequencies and used realistic LP neuron waveforms applied at different cycle frequencies. We found that the proctolin-activated current is more complex than previously determined and contains an inactivating component. Using a computational model, we explored the possible role of this inactivating component and show that its frequency dependence can produce state-dependent effects at the circuit level.Among the best studied neuropeptide modulators of the pyloric circuit is proctolin, which activates ron type , includiCancer borealis saline contained the following: 440 mm NaCl, 26 mm MgCl2, 13 mm CaCl2, 11 mm KCl, 10 mm Tris base, and 5 mm maleic acid, buffered to pH 7.4–7.5. Custom synthesized Proctolin  was dissolved in distilled water and stored as 10−3m aliquots at −20°C. Immediately before usage, proctolin stock was diluted in saline to a final concentration of 10−6m. To prevent neurons from spiking during voltage-clamp experiments, we added 10−7m tetrodotoxin , stored as 10−4m stock solution in distilled water at 4°C, to the saline to block voltage-dependent Na+ currents. In some experiments, we added 2 × 10−4m Cd2+ to the saline to block Ca2+ channels  were anesthetized by placing them in ice for at least 30 min. Their STNS was dissected as described previously (lvn). Extracellular electrodes were connected to a differential AC amplifier . LP was identified by matching its intracellularly recorded activity to the extracellularly recorded pyloric rhythm . Intracellular signals were amplified , and all recordings digitized at 5 kHz  and recorded with Clampex 10.6 (Molecular Devices). Voltage-clamp waveforms were created with MATLAB  and delivered via Clampex.We used the two-electrode voltage-clamp technique to measure proctolin-activated currents in the soma of the LP neuron. Electrodes were pulled from borosilicate capillaries with filament and filled with 0.6 −7m TTX saline to block action potentials, which prevents transmitter release from the terminals of modulatory projection neurons.To measure the proctolin-activated currents, we removed all intrinsic neuromodulators by transecting the stomatogastric nerve  and/or by perfusing the STG with 10IProc) were calculated as the difference between total currents measured in normal saline and in the presence of 10−6m proctolin  ramp] and back to −80 mV [negative (–) ramp] with different slopes (2+ as done previously (2+ with 200 μm Cd2+ (Sigma-Aldrich), added to the saline both in control and in the presence of proctolin.To measure the t slopes D, left. t slopes , and asseviously , we blocI-V relationship, in some experiments we used the same voltage range and slopes as before but held the voltage at +20 mV between the positive and negative ramps. Additionally, these ramp-and-hold waveforms were repeated until the I-V relationships reached a steady state. Thus, in these experiments the positive and negative ramps each contributed to 25% of one stimulus cycle; after the positive ramp, the voltage was held at +20 mV for 25% of the cycle, and after the negative ramp at −80 mV for 25% of the cycle, and this waveform scaled with ramp slope . Then, we subtracted the currents measured in control condition from the currents measured in the presence of proctolin to get the proctolin currents IProc. We averaged the last three stimulation cycles for the ramp stimulations, and the last five cycles for the ramp-and-hold stimulations. We fitted the averaged IProc separately for the positive ramps and negative ramps with a logistic equation:V is the membrane potential, g the maximal conductance, revE the reversal potential, ½V the half-maximal activation voltage, and Imax) and the corresponding voltage (VImax) at Imax at different slopes : 50, 100, 200, 400. Data for VImax failed normality and equal variance tests. Download Figure 2-1, XLSX file.Two-way RM ANOVA for initial state 10.1523/ENEURO.0338-21.2021.f2-2Extended Data Figure 2-2Cii. Download Figure 2-2, XLSX file.Data for panel IProc across the 30 ramp-and-hold sweeps with an exponential decay function:t is time and IProc (t0) and IProc (∞) are, respectively, the initial and steady state value of IProc.To calculate the time constant for the slow inactivation, we fitted the normalized, integrated IProc acquired with the realistic waveform stimulations, the current was baseline subtracted and filtered . Baseline current was measured as the average current in the 2 s before the start of stimulation. IProc was averaged for the last five cycles of stimulation, and we obtained Imax as well as the phase of Imax with LP trough potential as reference.To measure Imax between normal and Cd2+- saline we used two-way ANOVA. We used Tukey’s post hoc test for all pairwise comparisons. Data passed all tests for normality (Shapiro–Wilk) and equal variance (Levene’s test) unless noted otherwise. Significance level is α = 0.05. Results of statistical tests and raw data are provided in Extended Data All statistical analyses were performed in SigmaPlot 12.0 (Systat Software). For parameters obtained from ramp and ramp-and-hold stimulations, we used two-way repeated measures ANOVA (RM ANOVA) to test for effects of ramp slope , ramp direction , and interaction between the two factors. For the maximum inward current and phase of the maximum inward current obtained from realistic waveform stimulations, as well as the ratio of slow inactivation across ramps, we used one-way RM ANOVA. We compared time constants of slow inactivation across ramps with one-way ANOVA on ranks. To compare changes in IMI. Here, the steady-state IProc was modeled as a sum of two currents, the persistent current IMI and a transient current IMI-T:mMI, mMI-T and hMI-T—denoted as x) obeyed standard Hodgkin–Huxley style kinetics:The previously identified persistent current activated by proctolin has been found to be the same current as the persistent inward current activated by other modulators  and thusIMI-T as a (putative) calcium current using the Goldman–Hodgkin–Katz formalism:PCa is the total permeability of the current, mCa and hCa are activation and inactivation variables obeying vol is the volume of the microdomain influencing the current, F is Faraday’s constant, R is the universal gas constant and T is temperature. [Ca] is the calcium concentration outside (out) and inside (in) the cell. The internal calcium concentration obeyed the equationCaI denotes the total calcium current flowing into the cell  and [Ca]∞ denotes the steady state calcium concentration inside the cell, and P1 is the maximal per cluster permeability of CaI and P is the total permeability over all clusters of interest (IMI-T(Ca) were PCa = 0.014 cm/s, T = 283.15 K, [Ca]out = 13 mm, P1 = 1.1675 μm3/s, P = 0.0369 cm/s, vol = 6.49 μm3, [Ca]∞ = 0.02 mm, h∞([Ca]) = 1/(1+([Ca]/0.015)4) and τCa = 12 s.The parameters for the model are provided in interest . The parIleak, IA, Ih, ICa, IK(Ca), IMI, and IMI-T. Intracellular calcium accumulation was tracked in the soma/neurite compartment according to Ileak, INa, and IK. Except for ICa, all currents were modeled in the standard Hodgkin–Huxley formalism as:X denotes the current name, xE is the reversal potential and xm and xh respectively denote the current activation and inactivation  were set to 2) was set to 0.795 (axon) and 3.183 (soma/neurite). The parameters of ICa were PCa = 0.00187 nm/ms, T = 283.15 K, [Ca]out = 13 mm, P1 = 1.168 μm3/s, P = 0.0467 nm/ms, vol = 6.49 μm3. The equations for the ionic currents are given in An LP inspired model was adapted from  obeying  with app2 and a reversal potential of −80 mV. The model was then manually tuned to produce an LP-like voltage waveform.Inhibitory synaptic input was modeled as a symmetric triangular conductance waveform with a duty cycle of 0.5, a maximum value of 1 μS/mmIMI-T in our model LP neuron. This shift was estimated by adjusting the model for the steady state ramp-and-hold to respond as if IMI and IMI-T were expressed in a compartment ∼0.3 length constants away from the soma, rather than in the soma (fitting not shown). All simulations of the model LP neuron were done with these shifted values  and instantaneous spike frequency . Simulations were done in the Python NEURON (version 7.7.2) environment , which had a similar appearance for all ramp slopes (Imax) of IProc was larger on the positive ramp than on the negative ramp. Furthermore, the magnitude of the current was sensitive to the slope of the positive ramp, with larger slopes eliciting larger currents . For the negative ramp, Imax was mostly independent of slope value, except for the largest slope of 400 mV/s. Additionally, the voltage (VImax) at which Imax was attained was more depolarized for larger slopes on the positive ramp, and less so on the negative ramp (IProc includes both a persistent and a transient (inactivating) component. Both components activated with the positive ramp, but the negative ramp elicited mostly the persistent component because, by this time, the other component had inactivated. The presence of these two components would explain (1) the difference between the current amplitudes on the positive and negative ramps; (2) the slope dependence of the current amplitude on the positive ramps; (3) the fact that the largest negative ramp slope also revealed a larger current compared with other negative ramps, likely because of incomplete inactivation of the transient component; and (4) the shift of VImax to more depolarized values for larger slopes because with these more depolarized voltages are reached in a shorter amount of time, before complete inactivation of the transient component.With both positive and negative voltage ramps, proctolin elicited a voltage-gated inward current (p slopes A. Howeveive ramp . TogetheIProc measured with negative ramps was (mostly) independent of ramp slope and qualitatively matched the previously described IMI  component as IMI-T. Thus, the negative ramp current consists primarily of IMI, whereas the positive ramp current consists of IMI + IMI-T.The component of ibed IMI . TherefoIProc would be at some steady-state level of inactivation. To measure the steady-state levels of IMI and IMI-T, we applied symmetric ramp-and-hold stimuli repetitively for 30 cycles in a separate set of experiments : 100, 200, 400. Missing N: 200 mV/s: 2; 400 mV/s: 2. Download Figure 3-1, XLSX file.One-way RM ANOVA for the steady/initial state ratios of I10.1523/ENEURO.0338-21.2021.f3-2Extended Data Figure 3-2Figure 3-2, XLSX file.One-way ANOVA on ranks for the time constant of the slow inactivation. α = 0.05. Slope (in mV/s): 100, 200, 400. Missing N: 200 mV/s: 2; 400 mV/s: 2. Download 10.1523/ENEURO.0338-21.2021.f3-3Extended Data Figure 3-3C, D. Download Figure 3-3, XLSX file.Data for panels A, initial and steady state sweeps of the same experiment in B, quantification of the levels of slow inactivation in C and of the time course in D). We refer to the response to the first depolarizing ramp-and-hold stimulus in each run as the initial state, and to the average of the last five responses as steady state  as in the triangular ramp experiments without a depolarized hold interval. Generally, the steady-state I-V curves showed the same slope and directional dependencies as in the simple ramp currents described in Imax and VImax values at steady state on the negative ramp no longer showed slope dependence, probably because this difference was due to incomplete inactivation of the transient component with the triangular ramps.We explored the effect of ramp slopes at steady state . α = 0.05. Direction: positive or negative. Slope (in mV/s): 100, 200, 400. Download Figure 4-1, XLSX file.Two-way RM ANOVA for steady state 10.1523/ENEURO.0338-21.2021.f4-2Extended Data Figure 4-2C. Download Figure 4-2, XLSX file.Data for panel IProc has a slowly inactivating component, with a time constant of several seconds, that diminishes the total current activated and measured across frequently repeated stimuli until it reaches stable levels at steady state. Second, even at steady state, IProc has a persistent component (IMI) and a component that inactivates rapidly during depolarizing ramps (IMI-T).Together, these findings led to two conclusions. First, I-V curves revealed that IProc was most sensitive to the depolarizing slope. Because the pyloric rhythm operates over a large range of cycle periods , which corresponds to an increase in its depolarization slope. There was a small increase in the average IProc amplitude as cycle frequency was increased up to 2 Hz  was significantly smaller for the highest frequency tested  Ramps activate a current whose amplitude depends on the steepness and sign of the slope. (2) Realistic waveforms produce a current whose amplitude somewhat increases with increasing cycle frequencies (and thus larger positive slopes) up to 2 Hz but decreases at the highest frequency tested (4 Hz) in contrast to what is predicted from the ramp experiments. (3) The size of the current decreases gradually to a steady state over a timescale of several seconds when stimuli are repeated at frequencies consistent with pyloric cycle frequencies.Our findings so far led to three observations on IMI-T) be an inactivating current but could not be explained with a single persistent inward current that has been previously described  showed that the amplitude of the current did not consistently follow the same slope dependence as the current using the ramp protocols. Rather, the value of IProc decreased at the largest applied cycle frequency of 4 Hz (largest depolarization slope), which was the opposite of what we expected from the ramp experiments. As expected, when we simulated the simplest model that fit the ramp data with the LP voltage waveforms it produced the expected result that the current became consistently larger, not smaller, with increased cycle frequencies, and thus did not replicate the realistic waveform data (not shown). However, we noted that in our experiments the ramps spanned a voltage range (−80 to +20 mV) that was much larger than that of the realistic waveforms (−60 to −20 mV). It was therefore possible that the currents involved have different kinetics in the more restricted voltage range. Because the persistent current IMI has very fast activation kinetics  in the STG may have calcium permeability or even be calcium currents . Although we did not attempt to optimize the parameters of this model (many of which remain unmeasured), we found that this formalism could sufficiently explain how the current amplitude reduced with repetitive depolarization . α = 0.05. Directions: positive or negative. Slope (in mV/s): 40, 80, 160. Download Figure 7-1, XLSX file.Two-way RM ANOVA for steady state 10.1523/ENEURO.0338-21.2021.f7-2Extended Data Figure 7-2B. Download Figure 7-2, XLSX file.Data for panel IMI-T is a calcium current by repeating our ramp protocols with LP in saline containing 200 μm Cd2+, which blocks Ca2+ channels in the LP neuron . Only the largest positive slope of 400 mV/s resulted in a significantly larger Imax compared to the smallest slope of 50 mV/s  in Cd2+ saline. α = 0.05. Direction: positive or negative. Slope (in mV/s): 50, 100, 200, 400. Download Figure 8-1, XLSX file.Two-way RM ANOVA for initial state 10.1523/ENEURO.0338-21.2021.f8-2Extended Data Figure 8-22+. Failed normality and equal variance tests. Download Figure 8-2, XLSX file.Two-way ANOVA for the slopes of the linear fits across ramps with the same direction. α = 0.05. Direction: positive or negative. Saline: normal or Cd10.1523/ENEURO.0338-21.2021.f8-3Extended Data Figure 8-3IProc in initial and steady state in normal saline and Cd2+ saline. α = 0.05. State: initial or steady. Slope (in mV/s): 100, 200, 400. Download Figure 8-3, XLSX file.Two-way RM ANOVA for 10.1523/ENEURO.0338-21.2021.f8-4Extended Data Figure 8-4C. Download Figure 8-4, XLSX file.Data for panel 10.1523/ENEURO.0338-21.2021.f8-5Extended Data Figure 8-5Dii. Download Figure 8-5, XLSX file.Data for panel 10.1523/ENEURO.0338-21.2021.f8-6Extended Data Figure 8-6E. Download Figure 8-6, XLSX file.Data for Extended Data Figure 8-3IMI and IMI-T may differentially influence the LP bursting activity. Because we currently do not have an experimental method of separating the two components of IProc during the ongoing activity , we addressed this question in a computational model of the LP neuron  based on the model developed by IMI and IMI-T were adjusted to account for the spatial distance to the putative receptor location distal from the soma . Note that the lack of slope dependence with small amplitude stimulations ) and when it was not . We found that permeability to Ca2+ did not change the burst onset of the model neuron but shortened the burst duration so that the burst terminated before the arrival of the periodic inhibition. Consistent with the shorter burst, the number of spikes and the intra-burst spike frequency were both reduced, but there was no change in the burst structure in that the instantaneous spike frequency  decayed during each burst in both cases , when the model IProc was assumed to be permeable to Ca2+ (IMI-T(Ca), purple and pink traces), whereas the other ionic currents (which were not influenced by Ca2+ entry) were not affected (data not shown).To see how Cath cases Ai. To unIMI and IMI-T, we started with the model in 2+ permeability) at the cycle frequency of 1 Hz and tuned the levels of the modulatory currents to have seven spikes per burst. We refer to this as the reference model. We then changed the frequency of the periodic inhibitory synaptic input without changing the duty cycle, shape, or amplitude of this synapse. For simplicity, we only show the activity of the model neuron at two other frequencies, one lower (0.5 Hz) and one higher (1.66 Hz). Decreasing the cycle frequency slightly advanced the onset and end phases of the burst, and increased the number of spikes per burst, but did not affect the burst structure, in that finst followed the same decreasing trend within each burst  of the model neuron remained relatively constant for the duration of the burst. In contrast, in the IMI-T only case, both increasing and decreasing the cycle frequency shortened the duty cycle, but at 1.66 Hz, the burst was delayed in its onset, whereas at 0.5 Hz, it was advanced. In this case, the burst structure was also influenced by the change in cycle frequency. When cycle frequency was decreased, finst dropped dramatically within each burst (pink trace at 0.5 Hz), whereas it became somewhat more constant as cycle frequency was increased (pink trace at 1.66 Hz). The changes in the burst onset and end were reflected in the phase diagram of the model neuron (IMI only case (green) and the phases were more similar across cycle frequencies. In contrast, the shifts became more pronounced in the IMI-T only case (pink) and both the beginning and the end of the burst greatly shifted with increasing cycle frequency. Together, these modeling results indicate the ability of the two modulatory-activated currents to provide different influences on the burst structure, with IMI producing a consistent burst duration and strength across all cycle frequencies and IMI-T producing a dynamic change in burst structure across different cycle frequencies.In the l neuron Bii. In tOscillatory neuronal network activity can be stereotyped when conditions are stable  and can Frequency-dependent interactions in the nervous system are typically thought of as changes in neural activity that depend on the input frequency, as is the case with short-term synaptic plasticity. Neuromodulators can modify the frequency dependence of the effects of such inputs . HoweverIMI ). Large levels of IK(Ca), among other potassium currents, have been shown to prevent neurons from oscillating, even when pacemaker currents were enhanced . Such Ca2+ permeability of IMI-T would lead to the activation of IK(Ca) , will terminate the burst of spikes earlier than when only the persistent IMI is active (IMI-T effect on the follower LP neuron’s bursting activity is 2-fold: it boosts early excitability by inducing postinhibitory rebound (see below), and it provides a brake on prolonged excitability because of its transient nature and the recruitment of an outward current.Crustacean pyloric neurons are modulated by a host of endogenous peptides . Most ofIMI . Here, wal model . Howeverenhanced , and plaenhanced . Althoug(Ca) see Aii, withIMI is known to act as a pacemaker current in the pacemaker neurons of the pyloric network (IMI and IK(Ca)  . Howeveractivity . Althougolarized , leadingIMI-T, the total proctolin-elicited current (IMI + IMI-T) is more strongly activated by faster and positive-slope voltage ramps (IMI-T in the voltage range used for ramps (−80 to +20 mV) compared with those of the realistic waveforms  the current is strongly activated and generates a postinhibitory rebound but inactivates within the duration of a burst. Consequently, the burst terminates before the onset of the next cycle of inhibition , whereas IMI-T is much faster to activate and also inactivates rapidly. It is therefore unlikely that IMI-T is the same current as ITrans-LTS, but it is possible that IMI-T is the same as the fast transient current observed, but not further characterized, by ITrans-LTS, enables postinhibitory rebound bursting. Such rebound bursting has been shown to be important in the activity of a number of neurons  can have vastly different effects on postsynaptic neurons. For example, a recent report shows that dopaminergic neurons in the substantia nigra generate robust postinhibitory rebound bursts following inhibition by striatal neurons, whose transmissions target both GABA-A and GABA-B receptors. In contrast, inhibition of the same neurons by globus pallidus neurons, which activate only GABA-A receptors, does not generate rebound activity, but just pauses ongoing spiking , and all seem to vary in activation and inactivation kinetics. It is not known, but remains possible, that all convergently respond to multiple neuromodulators like IMI does (IMI-T and IMI, and perhaps also ITrans-LTS, could all be isoforms of the same channel. It is well known that ion channel isoforms can be generated by various mechanisms, including alternative splicing (C. borealis and the lobster Homarus americanus for multiple ion channel types (Previous studies that examined peptide modulation of STG neurons were mostly done in the presence of Cablockers , which me cycles  could in seconds C. This iemselves . Our finting IMI , but somIMI does , 2001. Tsplicing  and genesplicing , and thasplicing . The isosplicing , or pH ssplicing . Similarel types . This is2+ permeability of IMI-T give this current the potential to widely influence the activity of a neuron. Since LP is the only follower neuron that synapses onto the pacemaker group, frequency dependent changes in LP activity could in turn feed back to the pacemakers and stabilize the pyloric rhythm at a preferred frequency. It remains to be shown if IMI-T can be activated by other neuropeptides, similar to IMI, and if and to what extent IMI-T is present in other neurons.Overall, the transient nature and resulting frequency dependence, and Ca10.1523/ENEURO.0338-21.2021.f9-1Extended Data Figure 9-1Bii. Download Figure 9-1, XLSX file.Data for panel


Nature Reviews Molecular Cell Biology 10.1038/s41580-020-0237-9, published online 16 April 2020.Correction to: In the supplementary information, the reference to Contreras et al. 2016 was removed (as it relates to a gene different from E2-2/TCF4), and ‘(TCF7L2)’ was deleted from the table. Misspellings in the author name Ruby Y. J. Huang and in the PyMT glossary definition were also corrected. These changes have been made to the HTML and PDF versions of the article.


These results suggest that radiomic signatures may improve survival outcome predictions in GBM thus creating a solid clinical tool for tailoring therapy in this population.Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adult patients with a median survival of around one year. Prediction of survival outcomes in GBM patients could represent a huge step in treatment personalization. The objective of this study was to develop machine learning (ML) algorithms for survival prediction of GBM patient. We identified a radiomic signature on a training-set composed of data from the 2019 BraTS challenge (210 patients) from MRI retrieved at diagnosis. Then, using this signature along with the age of the patients for training classification models, we obtained on test-sets AUCs of 0.85, 0.74 and 0.58  for survival at 9-, 12- and 15-months, respectively. This signature was then validated on an independent cohort of 116 GBM patients with confirmed disease relapse for the prediction of patients surviving less or more than the median OS of 22 months. Our model insured an AUC of 0.71 (0.65 on train). The Kaplan–Meier method showed significant OS difference between groups (log-rank  The stan status . On the n status ,9,10,11,Magnetic resonance imaging (MRI) is the key radiological test used by neuro-oncologists for the evaluation and follow-up of brain tumors . It has To our knowledge, the robustness and generalization error of radiomics signatures have never been evaluated on different datasets. In this study, machine learning (ML) models for survival prediction of GBM patients from the training dataset of the 2019 BraTS challenge were developed. Based on these models, we identified a radiomic signature to validate a completely independent cohort of GBM patients.Data from the train dataset of the 2019 BraTS challenge ,24,25 weFor every patient, the dataset contained: the pre- (T1) and post-contrast T1-weighted (T1ce), the T2-weighted and fluid attenuated inversion recovery 3D MR images along with one ground truth mask containing 3 segmentation labels, covering 3 different anatomical parts of the tumor (the necrotic (NCR) and non-enhancing (NET) tumor core, the peri-tumoral edema (ED) and the enhancing tumor (ET)).This cohort was composed of 116 patients with histologically confirmed GBM . All thMR acquisitions were all performed on 2 imaging machines (MRI) from the same manufacturer : Optima MR450w 1.5T and Discovery MR750w 3T. MRI data included a post-contrast  three-dimensional T1-weighted Fast Spoiled Gradient Recalled (FSPGR) acquisition (post-contrast 3DT1), post-contrast 3DT1, and fat-suppressed FLAIR images. MR images were only used as inputs of the radiomics classifier. To ensure image quality, neuro-radiologists analyzed all the available imaging sequences. The whole pre-processing and post-processing pipelines are summarized in The nn-Unet  frameworOn both cohorts, radiomics were extracted from the two sub-masks (NCR/NET/ET and ED) on the T1ce and T2-FLAIR MRI modalities using the Python library Pyradiomics . The subSeveral ML algorithms were evaluated to process the BraTS cohort along 3 binary classification tasks: surviving less or more than 9, 12, and 15 months, respectively. GBM patients of the BraTS cohort were divided into train and test datasets. The proportion of each class in every classification model was kept in the train and test datasets. Cross-validations (CV = 5) were applied on the train datasets to avoid overfitting and to find the best feature-scaling method, classifier and hyper-parameter. Then, the best ML model found was trained on the overall train-set and evaluated on the test-set. Using the Scikit-learn Python library, 3 scaling methods  and 7 classifiers  were tested. Performances on the cross-validations were assessed using mean AUC over the 5 folds. In addition, due to the high dimensionality of the data, a feature selection using only the train-sets was imposed  to be then validated on the test-sets. Only features with a concordance-index (c-index) with an OS superior to the threshold were kept. Afterwards, the correlation between all the remaining variables was measured; if it was superior to a second threshold, the one with the lowest c-index was removed. The two threshold values were estimated by cross-validation. Due to the imbalanced classes in the 9- and 15-month models , a resamTo validate a potential radiomic signature for the survival prediction of GBM patients, the features selected on the BraTS cohort were used to classify the patients of the validation cohort into two groups: survival < or > 50th percentile in terms of OS (median of 22 months). Only the features selected in at least two models trained on the BraTS cohort were retained. Then, the same data processing was applied to the validation cohort . The c-index and correlation thresholds were retained to select the most appropriate signature.OS was calculated as the number of days between the diagnosis of GBM and death. Concordance index (C-index)  was usedSeveral ML algorithms were tested for each model. On the cross-validation, an RFT (random forest) obtained the best score for the 9- and 12-months classification models with a mean AUC of 0.75 ± 0.04 (±1 standard deviation) and 0.74 ± 0.14, respectively. For the 15-months model, a logistic regression had a mean AUC value of 0.69 ± 0.09. These best models on the cross-validations had an AUC on the test-sets of 0.85 (0.92 on the train-set) for the 9-months model, 0.74 (0.88) for 12-months and 0.58 (0.75) for 15 months. Precision and recall for each class of each classification model are provided in p = 0.05) with well-separated Kaplan–Meier curves (p = 0.04 for 12 months and log-rank p < 0.005 for 9 months). The class “before 12 months” had a survival probability of 0.29 at 12 months while the class “beyond 12 months” had a probability of 0.71. The results on the 9-months model were more significant with survival probabilities of 0.33 and 0.77 for “before 9 months” and “beyond 9 months”, respectively. These probabilities went down to 0 versus 0.37 for the same classes.Although the 15-months model had a low AUC value, it has succeeded to stratify GBM patients into two significantly different populations on the test-set (log-rank r curves . The surp = 0.05). Among the nine pre-selected features, five were kept in the final model of the validation cohort. The age, sphericity, and the NCT/NET/ET sub-mask appear to be of utmost importance in the prediction of OS within both our cohorts . The Kaplan–Meier curve  showed t cohorts . Models  cohorts  and 22-  cohorts  are avai cohorts .In this study, ML algorithms based on radiomics from MR imaging were designed for the survival prediction of GBM patients. A total of 448 radiomics from T1ce and FLAIR images were extracted from two independent cohorts of GBM patients. A radiomic signature was identified on the first cohort of 210 patients, arisen from the train dataset of the 2019 BraTS challenge. Then, the robustness of this signature for survival prediction of GBM was tested on a validation cohort composed of 116 patients. The results of this study suggest that these models can provide some valuable insights into the OS of GBM patients. The novel classification models successfully stratified patients into two significantly distinct populations, thus suggesting a possible role in the identification of GBM phenotypes. This classification reflects the aggressiveness of some phenotypes and might permit an optimal selection of therapy with personalized management based on the patient and tumor characteristics.Survival analysis was performed at three different endpoints: 9, 12, and 15 months on the 2019 BraTS challenge. Based on the features selection, a radiomic signature of nine features were identified including eight radiomics in addition to age. Using ML algorithms, the signature successfully stratified two unique populations at every endpoint. For instance, Chen et al.  reportedIDH wild-type (WT) GBM [The new 2021 World Health Organization (WHO) classification of central nervous system tumors recognizes molecular evaluation as a critical step in their classification. Mutation status of the IDH1 (isocitrate dehydrogenase), IDH2, and the co-deletion of 1p/19q are crucial for diagnosis, prognosis and prediction of chemotherapy benefit . Despite(WT) GBM . To furt(WT) GBM . Future (WT) GBM , transcr(WT) GBM , DCE-MRI(WT) GBM , blood d(WT) GBM  or diffu(WT) GBM  have alrOne relatively major limitation of this paper is the difference in the median OS of the two populations . Nevertheless, the BRATS population was largely heterogeneous due to the multicentricity without relevant data on the initial management of patients and the absence of a homogenous therapeutic strategy among the different cancer centers. While on the other hand, the validation cohort included GBM patients from a single expert center with a standard protocol (Stupp protocol) on initial diagnosis as well as on relapsing setting (bevacizumab) in addition to an adapted supportive care program for GBM, thus explaining the vast difference in OS between the two cohorts. Nevertheless, this limitation might represent an actual strength for this study since adapting the same features and survival model from the BRATS population have induced successful outcomes in the validation cohort similar to the training population, despite having two distinct populations with different characteristics.In conclusion, a signature of radiomics combined with age was identified which has successfully stratified a cohort of GBM patients into two distinct populations on 3 ML binary classification models for survival at 9,12 and 15 months. Additionally, this model significantly stratified two populations with different survival outcomes on a completely independent cohort of GBM patients. This study highlights the key role of radiomics for survival prediction and their potential of creating a daily clinical routine tool for decision-making and a more personalized approach.


Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a novel coronavirus that has caused a worldwide pandemic. The majority of medullary thyroid cancers present as a thyroid nodule. At the time of diagnosis, cervical lymph nodes and distant metastases are frequently detected.Here, we present a case of a 46-year-old man with coronavirus disease (COVID) pneumonia, who had persistently high serum procalcitonin levels despite normal C-reactive protein levels. The attending infectologist happened to be a colleague who spent some time, as part of her internal medicine rotation, in the Endocrine Ward and recalled that medullary thyroid cancer might be the cause. This led to the timely workup and treatment of the medullary cancer. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in an ongoing pandemic that has posed an extraordinary challenge for healthcare systems worldwide. Among other biomarkers, serum procalcitonin (PCT), C-reactive protein (CRP), ferritin, D-dimer, interleukin-6, and lactate dehydrogenase (LDH) are associated with disease severity and mortality. It is widely accepted that the increase in PCT level indicates the beginning of a critical phase of viral infection . In ChinMTC accounts for about 5% of all thyroid malignancies . Carcino1c level in the target range. Physical examination showed crepitation on the base of the right lung. Vital parameters were as follows: body temperature 36.7°C, blood pressure 134/94 mmHg, pulse rate 75/min, O2 saturation 97%–99%. On admission, CRP was 5.2 mg/L (reference range <5.0 mg/L) and procalcitonin (PCT) was 5.4 ng/ml (reference range <0.50 ng/ml). Quantitative and qualitative blood counts were normal. Chest computed tomography revealed irregularly shaped ground-glass opacities (GGOs) affecting the right lower lobe; lung parenchymal involvement was estimated to be 5%.A 46-year-old Caucasian man has been hospitalized in the Coronavirus Ward of the University of Debrecen Clinical Center in January 2021. He presented after 5 days of high fever, dry cough, nausea, and muscle pain. Both PCR and antigen tests confirmed SARS-CoV-2 infection. He has had a history of hypertension and type 2 diabetes known for 4 years and has been on a diet and metformin with recent HbA2 therapy. His diabetes was well-managed with diet and metformin. During his hospital stay, he received low-molecular weight heparin for the prophylaxis of thromboembolism. CRP remained low at 3.6 mg/L the day after admission, and no decline in the PCT level was detected (6.06 ng/ml). The consistently normal CRP with persistently high PCT levels could not be explained by infection. The infectologist (EV) who has seen endocrine patients during rotation in the internal medicine ward was seeking advice from an endocrinologist. Additional tests for MTC were performed; serum calcitonin and CEA levels were found to be 89.0 ng/L (reference range <11.8 ng/L) and 7.5 µg/L (reference range <3.4 µg/L), respectively.Antiviral treatment was started: favipiravir  1.6 g twice daily on day 1, followed by 600 mg twice daily for a total duration of 5 days. There was no need for OUltrasound of the neck region revealed normal sized thyroid with regular echostructure. Two small hypoechoic nodules, with largest diameters of 9 and 5 mm, were detected in the left lobe; no suspicious lymph nodes were seen in the neck region. Computed tomography (CT) demonstrated several 5–10-mm lymph node metastases on the left side of the neck, while no remarkable findings were seen in the chest. Fatty liver disease was suspected based on abdominal ultrasound.On-site frozen section histology confirmed MTC in a 14-mm lymph node removed from the region of the left jugular vein (level III). Total thyroidectomy was performed. Lymph nodes were excised from the central neck region (level VI) with central neck dissection (CND) and from the left lateral neck region  with modified radical neck dissection (MRND).Final histology revealed three malignant foci  in the left thyroid lobe. The right lobe was tumor-free. Tumor cells were positive for calcitonin, chromogranin-A, synaptophysin, and neural cell adhesion molecule (CD56) by immunohistochemistry Figure 1Follow-up values 4 weeks after surgery were as follows: serum calcitonin 11.3 ng/L , PCT 0.28 µg/L , CEA 1.8 µg/L , total calcium 2.51 mmol/L , phosphate 1.05 mmol/L . On 150 µg levothyroxine supplementation, thyroid-stimulating hormone (TSH) was 0.612 mU/L .The patient has been screened for clinical features of the MEN-2 syndrome, including 24-h urine metanephrines, serum calcium, ionized calcium, and parathormone measurements. His family history for MTC and MEN-2 components was negative. Rearranged during transfection (RET) mutational analysis did not reveal mutations in codons Cys609, Cys611, Cys618, Cys620, and Cys 634.The extreme burden of the COVID-19 epidemic results in overloaded medical systems and may lead to burn out of the overworked health care personnel. In the case presented here, the high PCT value detected on admission led to the hospitalization of the patient with mild COVID-19 infection and resulted in incidental detection of his MTC. On the day of admission, the attending infectologist happened to be a colleague who spent some time, as part of her internal medicine rotation, in the Endocrine Ward. When looking at the unusual combination of high PCT and near-normal CRP, she recalled that MTC might be the cause. Timely workup and treatment of the underlying MTC were successful Table 1.The 8- and 4-mm cancer foci on final histopathology corresponded to the 9- and 5-mm nodules detected by ultrasonography (US), while the 2-mm MTC focus was not detected by US. Fine-needle aspiration (FNA) was not performed, as our institutional protocol calls for thyroidectomy regardless of the FNA result if a serum calcitonin level above 5× upper limit of normal (ULN) in a patient with normal renal function is combined with CEA elevation. Of the imaging performed, the chest CT was part of the COVID workup on admission. The neck CT performed after the neck ultrasound may have been superfluous; although it revealed unexpected new information, it did not change the therapeutic decision. Furthermore, at CT, the largest lymph node was 10 mm, but at on-site histology, it was 14 mm. According to the detailed final histology report, the diameter of the metastasis was 12 mm in the 14-mm lymph node. All these point to certain inaccuracies in size measurement and call for improvement at our institution.One limitation of this case report is that we cannot provide a cutoff of PCT in detecting MTC in patients with COVID-19. Furthermore, there is still no agreement in the literature on the serum calcitonin cutoff to exclude MTC. In contrast to the 10-min half-life of calcitonin, PCT has a 22-35 hour half-life in serum (There are two ways how the coronavirus epidemic may interfere with thyroid care. On the one hand, COVID-19-related thyroid disorders can manifest as thyrotoxicosis, hypothyroidism, or non-thyroidal illness syndrome . On the SARS-CoV-2 infection and a disproportionately high level of PCT, relative to CRP, aided in the rapid detection and treatment of MTC. The endocrine training of the attending infectologist led to this unexpected benefit.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.Ethical review and approval were not required for the study on human participants in accordance with the local legislation and institutional requirements. The patient/participant provided a written informed consent to participate in this study.LS interpreted the patient data and did the literature review. LS and EN were major contributors in planning treatment and preparation of the manuscript. EV treated the COVID-19 symptoms and recognized the endocrinological disturbances. CM performed the pathological examination of tissue samples. FG and DK operated on the patient. ZB, FG, DK, and MB helped analyze patient data and draft the article. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Upper respiratory samples used to test for SARS-CoV-2 virus may be infectious and present a hazard during transport and testing. A buffer with the ability to inactivate SARS-CoV-2 at the time of sample collection could simplify and expand testing for COVID-19 to non-conventional settings.™ (Copan) as a possible transport and inactivation medium for downstream Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) testing to detect SARS-CoV-2. Inactivation of SARS-CoV-2 USA-WA1/2020 in eNAT and in diluted saliva was studied at different incubation times. The stability of viral RNA in eNAT was also evaluated for up to 7 days at room temperature (28°C), refrigerated conditions (4°C) and at 35°C.We evaluated a guanidium thiocyanate-based buffer, eNAT10 PFU/ml within a minute of incubation. When saliva was diluted 1:1 in eNAT, no cytopathic effect (CPE) on VeroE6 cells was observed, although SARS-CoV-2 RNA could be detected even after 30 min incubation and after two cell culture passages. A 1:2  dilution abrogated both CPE and detectable viral RNA after as little as 5 min incubation in eNAT. SARS-CoV-2 RNA from virus spiked at 5X the limit of detection remained positive up to 7 days of incubation in all tested conditions.SARS-COV-2 virus spiked directly in eNAT could be inactivated at >5.6 logeNAT and similar guanidinium thiocyanate-based media may be of value for transport, stabilization, and processing of clinical samples for RT-PCR based SARS-CoV-2 detection. These strict testing requirements limit the locations where SARS-CoV-2 assays can be safely performed. A transport buffer that inactivates SARS-CoV-2 while remaining suitable for RT-PCR detection assays would mitigate these safety threats and simplify widespread COVID-19 testing.The novel coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), the causative agent of Coronavirus induced disease 2019 (COVID19), emerged in Wuhan, China at the end of 2019 and has since infected almost one hundred fifty million people worldwide, causing a global pandemic . Simple https://www.copanusa.com/sample-collection-transport-processing/enat/) . 2021 Mar 5:2021.03.01.21252250. doi: 10.1101/2021.03.01.21252250. PMID: 33688663; PMCID: PMC7941634.3. The evaluation of stability of SARS-CoV-2 RNA in eNAT shows that eNAT does not interfere with qRT-PCR detection of viral RNA over a course of 7 days, but there is no data showing that eNAT improves the stability of viral RNA compared to a similar specimen without an inactivating substance. My experience is that raw saliva is stable for at least 7 days after heat inactivation without the need for preservatives, and this is documented in the SalivaDirect protocol.a. In the discussion the authors conclude, “eNAT, a guanidinium thiocyanate-based buffer can be used as a viral inactivation and preservation media for SARS-CoV-2 specimens.”b. I don’t think the data show that eNAT “preserves” the specimen. This may be a matter of nomenclature, where any media that doesn’t degrade the sample is considered to preserve the sample, but in my mind a preservative prevents normal degradation, and there is no evidence in this manuscript that eNAT reduces the normal degradation of RNA in saliva.c. My experience with heat-inactivated saliva is that the RNA is severely degraded but still detectable by RT-PCR. Any data or citation showing a reduction in degradation of RNA in saliva mixed with eNAT would be strong evidence that eNAT does stabilize/preserve the specimen.Minor Points1. From a practical standpoint, it is difficult to see how eNAT would be implemented in a saliva collection aid, which would need to come pre-aliquoted with a generous amount of eNAT to account for the variable amount of saliva collected. This does not affect the findings of the manuscript, but I am curious how the authors envision the use of this product.2. There is only a brief description of the Xpert Xpress SARS-CoV-2 test. Since this test was designed and validated for upper respiratory specimen such as nasopharyngeal swabs but is being applied to saliva specimen perhaps a more detailed explanation is appropriate. For instance, from what I could gather the volume of sample loaded into the Xpert Xpress cartridge is ~300uL. This is a significant deviation from standard RT-PCR protocols which use on the order of 20uL total for a single reaction.a. The authors only report results for the N2 target gene in the manuscript. This is certainly adequate to monitor for the presence of viral RNA, but is there a reason the results for the E target were omitted? Was there any interference from the eNAT on the amplification of the E target?**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, AttachmentPONE-D-21-8255.pdfSubmitted filename: Click here for additional data file. 18 May 2021Reviewer #1: Banik et. al. describes the use of a commercially available buffer to inactivate SARS-CoV-2 virus in saliva sample which is compatible with downstream RT-PCR applications. The research question is clear, and the methods used and results are adequate to answer the research question. The paper is well written in general. There are some minor comments Banik et. al. describes the use of a commercially available buffer to inactivate SARS-CoV-2virus in saliva sample which is compatible with downstream RT-PCR applications. Theresearch question is clear, and the methods used and results are adequate to answer theresearch question. The paper is well written in general.Question:Q: How common is saliva swab/sample being used for SARS-CoV-2 detection compared tonasal swab? Have you tested eNAT inactivation on nasal swab which is commonly used?https://doi.org/10.1101/2021.03.03.21251172), we demonstrated that a saliva swab in eNAT buffer harbors more virions and thus provides a higher detection rate compared to a nasal swab. In this study, we have demonstrated that SARS-CoV-2 spiked directly into eNAT transport media, which simulates any swab specimen in the media, completely inactivates SARS-CoV-2 spiked in at a concentration as high as 4x105 PFU/ml, although a nasal swab was specifically not tested.Ans: Saliva is a commonly used and popular non-invasive sample type for detection of SARS-CoV-2 from patients. In another related study, which is currently under review in a different journal and published in the preprint journal MedRXIV .Q: Line 40: instead of “after being opened” it is better to say “during processing”Ans: corrected as suggested in the revised manuscript (Line 42).Q: Line 61: instead of “be used”, it is better to use “help”Ans: corrected as suggested in the revised manuscript (Line 63).Q: Line 68 and 127 Use “SARS-CoV-2” negative instead of “COVID-19”Ans: corrected as suggested in the revised manuscript (Lines 70 and 129).Q: Line 82, 87, 95, 124, 128, 153, 175 etc.: change “X” to “´×”Ans: corrected as suggested throughout in the revised manuscript, where concentrations are mentioned.Q: Line 103: Please add the MWCO for the Slide-A-LyzerAns: The 10K MWCO is now added in the revised manuscript (Line 105).Q: Line 161, 162: change cell “lines” to “line”Ans: corrected as suggested throughout the revised manuscript where cell line is mentioned.Q: Table 1: Please add the MWCO for “Amicon Ultra 4mL” and Slide A-LyzerAns: Amicon ultra 100 KDa and 10K MWCO for Slide A-lyzer is now included in the Table 1 of revised manuscript. Q:Table 2 and 3: “Tissue” to “tissue”Ans: corrected as suggested in the revised manuscript where tissue culture media was mentioned (Table 2 and 3).Q: Table 2 and 3: I suggest centre the text in the tableAns: corrected as suggested in the revised manuscript (Table 2 and 3).Q: Table 2 and 3: “tested by both culture and TCID50” to “tested by both CPE and TCID50”Ans: corrected as suggested in the revised manuscript (Table 2 and 3).Q: Line 217: define “LOD”Ans: LOD is the limit of detection of the assay used in this study. A previous publication on the Xpert Xpress SARS-CoV-2 test defined and established the LOD , which was used as a reference, in our study. Q: Statistical analysisWhat’s the sample size in Figure 1? If the sample size is small and data is not normallydistributed, a non-parametric test  is more appropriate.What post hoc test was used on top of ANOVA ? Please provide the information in the manuscript or perform post-hoc test is it hasn’t been done.Ans: Ordinary One-way ANOVA with Tukey multiple comparison analysis was done using Graphpad Prism. This information is included in the revised manuscript.  Reviewer #2: This manuscript demonstrates eNAT transport buffer inactivates infectious SARS-CoV-2 virus in contrived swab and saliva samples. The authors also quantify SARS-CoV-2 RNA stability in eNAT for various lengths of time and at different temperatures. Overall the manuscript is technically sound and the data support the conclusions. However, not all data described is presented by the authors — the manuscript will be ready for publication following clarification of the following points:Q:* Lines 116-119 and 251-252 refer to data not shown in this manuscript, which is not permitted by PLoS journals. Please remove this text or include the referenced data.Ans: Data was written as a statement in the methods section (line 121) thus, ‘data not shown’ is now removed from the discussion section of the revised manuscript (Line 259).Q* Lines 123-124 of the Methods state: “For the first approach, 100µl of the SARS-CoV-2 virus culture (8X106 PFU/ml) was added to 2ml of eNAT”. However, lines 174-174 of the Results state: “The virus was added directly to eNAT at the final concentration of 8X105 PFU/ml”. If 100ul of 8e6 PFU/mL was added to 2mL, the final concentration would be ~4e5 PFU/mL. Please clarify.Ans: The reviewer is correct, and we have corrected this oversight in the revised manuscript (Lines 181 and 185). We thank the reviewer for catching this error.Q:* Lines 185-186 describe the “>5.6 log10 PFU/ml reduction in viral load” following mock swab inactivation with eNAT. 5.6 log10 PFU/ml is roughly equivalent to 4e5 PFU/mL so this statement is accurate if this was the viral treatment titer (see previous point). However, line 199 repeats that the inactivation efficiency is >5.6 log10 PFU/mL for virus-spiked saliva, even though the authors used a less concentrated viral stock to generate the contrived saliva samples . How can the inactivation efficiency be the same for mock swab and saliva samples when the titer of the mock saliva samples was lower? Please update the statement about saliva sample inactivation .Ans: The statement has been updated in the revised manuscript with the accurate numbers (Line 205).Q:* Lines 245-247 state: “Both dialysis and filtration using Pierce DRSC spin columns resulted in complete removal of basal cytotoxicity from eNAT and did not result in any viral loss”. Additionally, lines 135-137 state: “Positive control samples were also assessed before and after DRSC purification to determine viral loss during this process.” However, this manuscript does not present data demonstrating viral titers are equivalent pre and post dialysis/filtration. Please amend the claim in 245-247 and remove lines 135-137 .Ans: The statement regarding the positive control is accurate and is presented in the revised Table 1 of the revised manuscript. Reviewer #3: The manuscript is technically sound, with appropriate controls. It is well written and clear, easy to understand. The results have useful implicatios for sample testing as explained in the intro/discussion.A few minor points:Q: 1. In intro, lines 38-39: suggest define sars-cov-2 and covid19 upon first use.Ans: SARS-CoV-2 and COVID-19 are now defined briefly in the introduction of the revised manuscript (Lines 37-40).Q:2. lines 135-137 explain that a positive control was included to show no loss from DRSC , can these data be added/explained to show there's no loss? .Ans: The data has now been added to Table 1 of the revised manuscript.Q: 3. method lines 148-150 explain the RT-PCR assays, but can it be more clearly stated in results what is being measured in each section . The PCR results are used to show inactivation, for example in Table 2, so negative on PCR means virus is inactivated, but one would expect RNA to still be present (which is shown later in stability section), so different things are being measured via RT-PCR in the different sections. But if this can be briefly, clearly described that might be beneficial for some readers.Ans: As correctly stated by the reviewer, yes in this experiment we have used RT-PCR as a measure for viral growth to study inactivation. After two passages on Vero E6 cells, viral RNA is not expected to be present since there are no replicative viruses present, if the virus is inactivated. If there are very few replicative virions present, then viral RNA will be detected at very late Ct values, indicating either incomplete inactivation, which was what we observed in our study for initial time points (0 min and 1 min). In contrast, our stability study examined virus that was spiked into the eNAT buffer and was tested by RT-PCR directly, without viral propagation. Here, one would expect to see unchanged Ct values at different conditions and time if the viral stability is preserved by the eNAT buffer. We have included part of this explanation in the revised manuscript to make it clear (Lines 150-152). Reviewer #4: Review SummaryThe manuscript titled, “Inactivation of SARS-CoV-2 virus in saliva using a guanidium based transport medium suitable for RT-PCR diagnostic assays” is concise and well-written. It clearly demonstrates the effectiveness of eNAT, a guanidium thiocyanate containing buffer, at rapidly inactivating live SARS-CoV-2 virus when used at a ratio of 2:1 with saliva or as a transport medium for swab specimen. eNAT supported detection of low concentrations of SARS-CoV-2 RNA for at least 7 days at a range of storage temperatures with almost no loss of sensitivity. Q: The manuscript suggests in passing that the eNAT buffer also preserves the specimen, although this is not explicitly addressed by the data. Ans: We have revised the phrase to include stability and not preservation. The stability of the RNA in eNAT is demonstrated conclusively.Q: There are some minor inconsistencies in the reported concentrations of SARS-CoV-2 virus used in the manuscript, but they do not affect the conclusions of the paper. Overall, the research presented here is of use to the public health and clinical testing communities and represents a useful addition to the available arsenal of specimen collection mechanisms, but the scope of the findings is necessarily narrow.Q1. Is the manuscript technically sound, and do the data support the conclusions?Partly: The manuscript is technically sound, but the reported viral concentrations and dilutions don’t totally add up. The data support the main conclusion that eNAT can inactivate SARS-CoV-2 in saliva at a ratio of 1:2  in as little as 5 minutes.Ans: we agree with the reviewer there was a mistake with the concentrations. But we have corrected that in the revised manuscript. Q2. Has the statistical analysis been performed appropriately and rigorously?Yes.Q3. Have the authors made all data underlying the findings in their manuscript fully available?Partly: The data from the CPE and TCID50 dilution series are only presented in a final summary. I don’t know if it is customary to present the entirety of this type of data, but I have seen it presented in other manuscripts.Q4. Is the manuscript presented in an intelligible fashion and written in standard English?Yes, the manuscript is well written and easy to read.Ans: none needed to Q2-Q4. We thank the reviewer for his/her supportive comments.Important PointsQ: 1. In the ”Inactivation of SARS-CoV-2 with eNAT” results section it states that SARS-CoV-2 is diluted to a final concentration of 8x10^5 PFU/mL in eNAT. The methods section describes diluting 100uL of 8x10^6 PFU/mL in 2mL of eNAT which should give you a final concentration of ~4x10^5 PFU/mL.a. The results describe a “100uL positive control containing 8x10^6 PFU/mL of SARS-CoV-2” but the methods suggest that this control was further diluted in 2mL of DMEM to the same concentration as the eNAT test samples (~4x10^5 PFU/mL).Ans: The reviewer is correct, and we have corrected this oversight in the revised manuscript (Lines 181 and 185).Q: b. Ideally the concentrations of virus should be reported consistently throughout the manuscript as either the final diluted concentration or the stock concentration into a known dilution volume.Ans: Corrected to maintain consistency throughout the manuscript in the revised copy. Q: c. The 8x10^5 PFU/mL diluted concentration does not make sense given the stock concentration and dilution volumes listedAns: we have corrected this oversight in the revised manuscript (Line 181).Q: 2. Although the SARS-CoV-2 titers used in the manuscript are reasonably high, it is not uncommon to see quantities of genomic fragments in saliva on the order of 10^8-10^10 copies/mL (~10% of cases) and >10^10 copies/mL in a smaller number of cases (~2%) based on the preprint below, using qRT-PCR. I see similar results using ddPCR for quantification. Although these PCR-based methods do not quantify infectious virions, is an inactivation efficacy >5.6 log10 PFU/ml an appropriate benchmark for SARS-CoV-2 inactivation of saliva samples?a. Yang Q, Saldi TK, Lasda E, Decker CJ, Paige CL, Muhlrad D, Gonzales PK, Fink MR, Tat KL, Hager CR, Davis JC, Ozeroff CD, Meyerson NR, Clark SK, Fattor WT, Gilchrist AR, Barbachano-Guerrero A, Worden-Sapper ER, Wu SS, Brisson GR, McQueen MB, Dowell RD, Leinwand L, Parker R, Sawyer SL. Just 2% of SARS-CoV-2-positive individuals carry 90% of the virus circulating in communities. medRxiv [Preprint]. 2021 Mar 5:2021.03.01.21252250. doi: 10.1101/2021.03.01.21252250. PMID: 33688663; PMCID: PMC7941634.Ans: We agree with the reviewer that a study with even higher concentrations of virus would have been ideal. However, the virus stock used in our manuscript represented that obtained from optimized culture methods followed by 10X concentration. With stocks as high as 8x10^6 PFU/ml, our study is really at or close to the upper limit of virus concentrations that are available. We have spoken to our colleagues who have similar observations. Line 259 does state that studies with even higher concentrations would have been preferable.Q: 3. The evaluation of stability of SARS-CoV-2 RNA in eNAT shows that eNAT does not interfere with qRT-PCR detection of viral RNA over a course of 7 days, but there is no data showing that eNAT improves the stability of viral RNA compared to a similar specimen without an inactivating substance. My experience is that raw saliva is stable for at least 7 days after heat inactivation without the need for preservatives, and this is documented in the SalivaDirect protocol.Ans: We agree with the reviewer that the virus in saliva in the absence of any preservatives is stable for at least 7 days, but not inactivated. Here we demonstrated that eNAT can be used both as an inactivating and stabilization buffer for saliva samples from SARS-CoV-2 infected patients even with low viral load. a. In the discussion the authors conclude, “eNAT, a guanidinium thiocyanate-based buffer can be used as a viral inactivation and preservation media for SARS-CoV-2 specimens.”Q: b. I don’t think the data show that eNAT “preserves” the specimen. This may be a matter of nomenclature, where any media that doesn’t degrade the sample is considered to preserve the sample, but in my mind a preservative prevents normal degradation, and there is no evidence in this manuscript that eNAT reduces the normal degradation of RNA in saliva.Ans: We have revised the phrase to include stability and not preservation (Line 32).Q: c. My experience with heat-inactivated saliva is that the RNA is severely degraded but still detectable by RT-PCR. Any data or citation showing a reduction in degradation of RNA in saliva mixed with eNAT would be strong evidence that eNAT does stabilize/preserve the specimen.Ans: To our knowledge, our study is the first to report that eNAT can stabilize and inactivate SARS-CoV-2 RNA in saliva samples. However, since eNAT buffer is a guanidium based buffer, studies have shown that the RNA is stable in guanidium based buffers . We have updated the revised manuscript to include these references in line 238.Minor PointsQ: 1. From a practical standpoint, it is difficult to see how eNAT would be implemented in a saliva collection aid, which would need to come pre-aliquoted with a generous amount of eNAT to account for the variable amount of saliva collected. This does not affect the findings of the manuscript, but I am curious how the authors envision the use of this product.https://doi.org/10.1101/2021.03.03.21251172). In that study, we added a swab of saliva to eNAT media, which showed comparative results as direct saliva. We also studied saliva added at 1:2 ratio in eNAT buffer, where a commercially available 2 ml eNAT media is added with 1 ml of saliva and showed better performance than nasal or oral swab. Thus, we do see the potential for a sample collection media which can keep the sample both stable and sterile for a period at different temperatures. Ans: eNAT is sold commercially for collection of upper respiratory specimens in 2ml or 3 ml aliquoted tubes with swabs by Copan inc. We used the same in another study where we evaluated different respiratory specimens from patients in different media Ans: Please see the above related answer to Q1. The application of Xpert Xpress SARS-CoV-2 test for different sample types is described in detail in another study published by some of the authors from this study, which is available as a pre-print in MedRXIV  on day 7 of 1:2 eNAT in saliva samples stored at 28°C.________________________________________1. Barra, G. B., T. H. Santa Rita, P. G. Mesquita, R. H. Jácomo and L. F. A. Nery (2021). "Overcoming Supply Shortage for SARS-CoV-2 Detection by RT-qPCR." Genes 12(1): 90.2. Loeffelholz, M. J., D. Alland, S. M. Butler-Wu, U. Pandey, C. F. Perno, A. Nava, K. C. Carroll, H. Mostafa, E. Davies, A. McEwan, J. L. Rakeman, R. C. Fowler, J. M. Pawlotsky, S. Fourati, S. Banik, P. P. Banada, S. Swaminathan, S. Chakravorty, R. W. Kwiatkowski, V. C. Chu, J. Kop, R. Gaur, M. L. Y. Sin, D. Nguyen, S. Singh, N. Zhang and D. H. Persing (2020). "Multicenter Evaluation of the Cepheid Xpert Xpress SARS-CoV-2 Test." J Clin Microbiol 58(8).AttachmenteNAT Response to Reviewer comments_PlosOne 5521.docxSubmitted filename: Click here for additional data file. 20 May 2021Inactivation of SARS-CoV-2 virus in saliva using a guanidium based transport medium suitable for RT-PCR diagnostic assays.PONE-D-21-08255R1Dear Dr. Banada,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Ruslan Kalendar, PhDAcademic EditorPLOS ONE 2 Jun 2021PONE-D-21-08255R1 Inactivation of SARS-CoV-2 virus in saliva using a guanidium based transport medium suitable for RT-PCR diagnostic assays. Dear Dr. Banada:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. 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Total hip arthroplasty (THA) with ceramic-on-ceramic (CoC) was created to minimise wear debris and aseptic loosening. A decade ago, a meta-analysis showed a 10-year survival rate of just 89%. Based on the excellent tribology of the current CoC, significant improvement of implant survivorship is expected. In patients younger than 60, we conducted a meta-analysis to assess 10-year survival and complications after using current primary CoC THA.PubMed, Scopus, EMBASE, Virtual Health Library, and Cochrane Library were used to scan for published trials that met the inclusion criteria until January 2019. The qualified studies were subjected to a systematic review and proportional analysis, and the randomised controlled trials (RCTs) were included in a comparison meta-analysis.Thirteen studies were included 156 findings. The total number of hips was 2278. Nine studies were cohort, and four were RCTs between ceramic and polyethylene cups. The analysis revealed an average age of 44 years (range 24–54). The 10-year survival 96% , aseptic loosening rate 0.516. , ceramic fracture rate 0.620  and squeaking rate 2.687 . A comparison meta-analysis revealed the risk ratio (RR) for revision was 0.27 , and for aseptic loosening 0.15 (0.03–0.70) favouring CoC, while RR for component fracture was 1.62  favouring the polyethylene.In patients under sixty, current CoC THAs are correlated with better 10-year outcomes than before and have high survivorship rates.Level of evidence: Level I. With longer life expectancy, total hip arthroplasties (THA) in young patients are likely to wear out faster with higher rates of aseptic loosening. While there is agreement on cementless fixation, there is no such agreement on articulating surfaces .Ceramic-on-ceramic (CoC) THAs have low friction and wear due to their mechanical and chemical properties and surface lubrication by joint fluid, resulting in less osteolysis than other bearing surfaces currently available , 3.Ceramics' brittleness, on the other hand, raises the possibility of component fracture. The replaced joint's squeaks are also a cause of concern. In the young and active patient, the best bearing surface for THA is still up for discussion –7. So, cThus, we performed a systemic review and meta-analysis of studies that reported 10-year survival based on the CoC bearing of the 3rd-and 4th-generation CoC THAs in patients younger than 60 to address their complications and survival rate.In Jan 2019, the study was conducted according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement (PRISMA) rules [Forte) and 4th-(Delta) generations with reported 10-year survival. Studies included near 10-year results or more of Biolox forte or delta, full text in English, prospective or retrospective, cohort or RCTs were included.Medical Subject Headings (MeSH) terms and free words were used, including ceramic  and hip arthroplasty . Two investigators reviewed each article independently to identify the publication relevance to the eligibility criteria included primary THA for patients < 60 years with CoC bearing surfaces of the 3rd-  , 11. It The included studies were pooled using the Mantel–Haenszel fixed-effects method (FEM) and the DerSimonia Laird random-effects method (REM). In the absence of significant heterogeneity, the FEM was considered; otherwise, the REM was considered.p value < 0.01) denoted heterogeneity among the studies.Cochran Q chi-square test: A statistically significant test (Q is the chi-squared statistic and df is its degrees of freedom.I-squared (I2) index, which is calculated as follows: Studies included were tested for heterogeneity using the following tests:I2 test, a Mantel-Haenszel random-effect was planned. The continuous outcomes were expressed as mean and standard error (SE) and 95% confidence limits (95% CI). The odds ratio (OR) with 95% CI was calculated for binary outcomes. Data for survival percentage (frequency) were pooled using the inverse-variance method and the Freeman-Tukey double arcsine transformation to calculate proportion before returning to the original scale for plotting.In the case of heterogeneity >50% at p value < 0.05 denoted statistical significance.Statistical analyses of the meta-analysis were performed using RevMan V.5.3 . A two-sided We identified a total of 156 studies, 23 duplicates were omitted. After application of our eligibility and exclusion criteria, we excluded 103 records. Due to the low quality of research according to NOS, we again excluded nine records. Again, eight records were excluded: due to the inaccessibility of English full texts. That leaves us with 13 records available for data extraction and quantitative synthesis. Figure 1These studies included four RCTs and nine cohort studies; five studies were prospective, four studies were retrospective, and all were published in peer-review journals between 2006 and 2018 –23. The In general, the methodological quality of all the cohort trials was low in bias risk. In terms of NOS, the mean value was 6.7 stars. Three studies got a 6-star rating , 16, 19,The proportional analysis was conducted for all 13 studies, and the comparison meta-analysis included four RCTs , 18, 20 survival The mean 10-year survivorship was 96% . The random-effects model was used due to considerable heterogeneity (I2 = 99%). The proportion plot at the end of ten years is shown in Fig. 10-year geneity I = 99%. TAseptic loosening rate was 0.516 . Table I2 = 22%); therefore, the fixed-effects model has used.The audible squeaking rate was 2.687% . There was considerable heterogeneity (I2 = 84.6%); therefore, the random-effects model was used.Component fracture rate was 0.62% . There was moderate heterogeneity (I2 = 22%); therefore, the fixed-effects model was used.Generation-specific complications; component fractures occurred in 11 cases out of 1339 hips in the 3rd-generation (0.8%) but decreased significantly to two cases out of 939 hips in the 4th-generation (0.2%) (p = 0.057). This was not the case when comparing the prevalence of squeaking, which was 2.9% in the 3rd-generation (40/1339 hips) and 2% in the 3rd-generation (19/939 hips), a statistically insignificant difference (p = 0.178).Risk Ratio (RR) of revision due to any cause was 0.27 , and for aseptic loosening was 0.15 ; both were in favour of CoC. Contrary to the results mentioned above, RR for component fractures was 1.62 , favouring the polyethylene group as is shown in Table Four RCTs compared the outcomes of THA between CoC and polyethylene bearings, including 1191 hips. The survival rate for CoC was 96% , and for the polyethylene was 92.4 . Hip replacement surgery is one of the most successful operations in all of medicine. Since the early 1960s, joint replacement surgical techniques and technology improvements have increased total hip replacement effectiveness , 25. AccTotal hip arthroplasty is becoming more common in younger patients around the world. American Academy of Orthopaedic Surgeons (AAOS) reported a 123% increase in THA rates in the 45–64 age group from 2000 to 2009 . YoungerA decade ago, a meta-analysis tested three bearings CoC, MoP, and metal on metal (MoM), when used in young patients, and concluded that ceramic articulations had the lowest survival among the bearing surfaces  with the highest survival rate for MoM articulations (95.4%) . HoweverCeramic-specific complications include ceramic fracture, squeaking, and aseptic loosening, have been linked to a higher failure of CoC implants than hard-on-soft implants as MoP . Early cP < 0.001), tested in 13 studies that included 2278 patients with the 3rd- and 4th-generation CoC bearings. This survival rate is similar to the patients' of 60 years or more, recorded to be from 95.6 [Our meta-analysis found some expected findings consistent with previously published studies, and it also produced some new data by combining the included studies. In the proportional analysis, the 10-year survival rate of CoC THA was recorded as 96% . The study strengths are the large patient sample size (2278 hips) and the ability to examine multiple risk factors. Besides, this study was able to combine multiple implants from various manufacturers. Finally, all the studies were published in the last fifteen years.One weakness is that most of the included studies were level-of-evidence 2–4 studies. Only four of the included 13 studies were level-1-study. An effort was made to perform a "best evidence meta-analysis," including studies with appropriate methodology. Not every study examined the same variables. Thus, for each of the subgroup analyses, there were a different number of studies included.Ceramic on ceramic-bearings in current THA has been associated with improved 10-year results and survival when used in patients less than 60 years than what was documented before. The improved manufacturing process of ceramic surfaces has a significant impact on these results.